Increasing evidence demonstrates that cancer cells from solid tumors are highly heterogeneous, but it has been challenging to identify and to isolate those extremely tumorigenic tumor-repopulating cells as surface marker methods have been proven to be unreliable and their effectiveness has been controversial. During the last funding cycle, we developed a novel mechanical method of selecting and growing a subpopulation of cancer cells from melanoma cells using a soft fibrin matrix and demonstrated that they are highly tumorigenic in wild-type syngeneic and even nonsyngeneic mice. In this competitive renewal, we propose to elucidate the underlying mechanisms of how forces regulate gene expression, which is crucial in elucidating force-induced differentiation of highly tumorigenic tumor-repopulating cells. Our preliminary results suggest that epigenetic changes of H3K9 methylation levels are a link between force and Sox2 expression in the tumor-repopulating cells. We further show that focal adhesion kinase (FAK) activity is high in self-renewing tumor-repopulating cells but low in invading tumor-repopulating cells in the soft 3D matrix, that FAK activity in the cytoplasm and H3K9 methylation in the nucleus is inversely associated, and that silencing FAK leads to H3K9 methylation and inhibition of self-renewal of tumor-repopulating cells. Importantly, a local force of physiologic magnitudes via integrins on the cell surface is shown to be able to unfold chromatin segments and induce gene expression, suggesting a direct force-propagating pathway for gene expression in a live cell. Built on these preliminary results, we propose 3 specific aims to elucidate mechanotransduction mechanisms in the living cell: Aim 1: To elucidate how a surface force regulates methylation of H3K9 and self-renewal gene expression in tumor- repopulating cells; Aim 2: To elucidate how FAK activity and H3K9 methylation are regulated in TRC growth; Aim 3: To determine how a surface force alters gene expression in the nucleus of a cell. Our preliminary results support the feasibility of all 3 aims. We will employ several bioengineering approaches to apply forces to the living cell, and combine FRET technology, 3D imaging, BAC (bacterial artificial chromosome) transgene techniques to visualize, to quantitate, and to map mechanotransduction processes in the cytoplasm and in the nucleus of the living cell. The long term goal is to understand how forces and mechanical microenvironment in the 3D matrix regulate expression of self-renewing genes and differentiation genes, fundamental in elucidating how highly tumorigenic tumor-repopulating cells sustain self-renewal in soft low-force microenvironment.